Shiana: Trial, Error, and Success

This past week I got my first taste of what it means to do real laboratory science. Every step must be double-checked for success and no pain should be spared to attain the materials needed for a project to yield optimum results. Unfortunately, ordering these materials have caused some small delays already because companies seem to be slower in the summer time delivering their products. We have found ways to continue to be productive, however.

My mentor Rae and I had both planned last week to begin the next step of our project by Monday, but we were held up because we noticed in our last experiment that the DNA concentration of Prx III we ended up with was much lower than normal. It was confusing at first because Rae had never gotten such consistently low concentrations before. To test the control plasmids we had rendered by conducting a smaller experiment (with one control ampicillin plate that grew and two amp. plates that did not), we had to conclude that the problem lay with the primers we had ordered. By asking around, Rae was able to locate another vector the lab used that always yielded good results in the past. In order to use this vector, however, we needed to design and order a new set of primers. These should come in by next Monday (though we had requested them by Thursday).

Meanwhile, for practice and experience I set about replicating the steps for creating the control Prx III plasmid I had watched Rae do last week. My digestion did not work the first time I tried it, which was frustrating especially since when I went over my procedure with Rae and showed her exactly what I had done, she could find nothing wrong with how I conducted the digestion. She gave me a few tips and helped supervise me the second time I did my digestion. When I checked my gel for my results, I found that only my plasmid had been cut and not my vector. The only thing that we could come up with that went wrong was that too much of the restriction enzymes NotI and EcoRI could have been added in, since both have a tendency to cling to the inside of a pipette tip because of the glycerol in it. There was no time to conduct the experiment a third time because it was already 5 P.M., so I left for the day.

The next day my mentor and I decided to alter digestion system volumes and I started right in on my project when I got into the lab (usually I am the first or second to arrive). This time, reading my gel was tricky because while my plasmid clearly worked, my vector lane had two bands that were very close together. Usually, you only expect to see either three bands if it didn’t work, or one if it did work. I chose to clean up the DNA in both lanes and continue on with my experiment to make sure if it worked or not, making two ligation systems with vector DNA from Rae and from my own experiment so I could compare them.

I was nervous the rest of the week for whether or not my experiment would work or not while I tried very carefully to follow my procedure notes accurately, and lo and behold! Patience and persistence paid off on Friday when I found out that all my trials had worked after all. This has boosted my confidence because now I know for sure that I am capable of conducting the rest of my experiment on my own.

Also, the journal club presentation by our guest speaker was very much enjoyable this week. He was quite involving and did not hesitate to tackle complicated concepts with us or dole out scientific jargon. Laughter, as always, was abundant and it was clear everyone had fun being together as a group and talking about their projects and research papers. I am very eager and look forward to my last three weeks of this internship, and in fact I have already discussed with my mentor possibilities for staying on in the lab for the rest of the summer. I love the whole atmosphere and feeling of actual contribution.